Protease Activated Receptors and Arthritis

International Journal of Molecular Sciences

Review Protease Activated Receptors and Arthritis

Flora Lucena and Jason J. McDougall *

Departments of Pharmacology and Anesthesia, Pain Management & Perioperative Medicine, Dalhousie University, 5850 College Street, Halifax, NS B3H 4R2, Canada; ﬂ[email protected] * Correspondence: [email protected]

Abstract: The catabolic and destructive activity of serine proteases in arthritic joints is well known; however, these enzymes can also signal pain and inﬂammation in joints. For example, thrombin, trypsin, tryptase, and neutrophil elastase cleave the extracellular N-terminus of a family of G protein- coupled receptors and the remaining tethered ligand sequence then binds to the same receptor to initiate a series of molecular signalling processes. These protease activated receptors (PARs) pervade multiple tissues and cells throughout joints where they have the potential to regulate joint homeostasis. Overall, joint PARs contribute to pain, inﬂammation, and structural integrity by altering vascular reactivity, nociceptor sensitivity, and tissue remodelling. This review highlights the therapeutic potential of targeting PARs to alleviate the pain and destructive nature of elevated proteases in various arthritic conditions.

Keywords: arthritis; inﬂammation; joint damage; proteases; pain

1. Introduction Musculoskeletal diseases comprise the most prevalent chronic pain conditions with Citation: Lucena, F.; McDougall, J.J. arthritides accounting for the majority of these disorders [1]. Although there are over Protease Activated Receptors and 100 different types of arthritis, the most commonly studied are inflammatory rheumatoid Arthritis. Int. J. Mol. Sci. 2021, 22, arthritis (RA) and degenerative osteoarthritis (OA). In RA, an individual’s immune system 9352. https://doi.org/10.3390/ is dysregulated and inflammatory cells begin to destroy the host joint tissues by releasing ijms22179352 chemical mediators into the joint. Autoantibodies such as rheumatoid factor and antibodies directed towards activated citrullinated proteins are also prominent features of RA which Academic Editor: Giovanni Tarantino contribute to the degenerative process and pain [2]. The RA synovium becomes hyperplastic and this pannus invades cartilage, bone, and menisci leading to altered biochemical Received: 21 July 2021 regulation and tissue damage. OA is characterized as an inappropriate healing response to Accepted: 26 August 2021 joint tissue injury whereby cartilage exhibits focal lesions, subchondral bone is dense and Published: 28 August 2021 fissured, osteophytes form, and the menisci can become calcified [3]. Intermittent synovitis occurs in some patients and joint inflammation is heterogeneous [4]. Pain and joint stiff- Publisher’s Note: MDPI stays neutral ness are common to all types of arthritis and limit an individual’s ability to move which with regard to jurisdictional claims in could put them at risk of other co-morbidities such as diabetes, obesity, or cardiovascular published maps and institutional affil- disease [5–7]. iations. The available treatments to manage joint diseases are currently very limited and have variable efficacy between patients. Pharmacological treatments for RA include the use of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, methotrexate, and biologics that inhibit cytokine reactivity [8]. With respect to OA, there are no disease Copyright: © 2021 by the authors. modifying drugs and treatments are restricted to symptom relief. Topical NSAIDs, ac- Licensee MDPI, Basel, Switzerland. etaminophen, serotonin-noradrenaline reuptake inhibitors, and opioids are typically used This article is an open access article to treat the disease [9]. All of the drug therapies have varying degrees of effectiveness and distributed under the terms and can produce undesirable side effects. The search for new drug targets that can control pain, conditions of the Creative Commons inflammation, and function is therefore an important area for ongoing arthritis research. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ The chemical mediators responsible for generating joint disease are still being defined 4.0/). but are known to include prostaglandins, neuropeptides, and cytokines [10]. Emerging

Int. J. Mol. Sci. 2021, 22, 9352. https://doi.org/10.3390/ijms22179352 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9352 2 of 16

evidence also indicates that proteolytic enzymes can also signal pain and inflammation in joints by cleaving a specific receptor family known as the protease activated receptors (PARs; Table1). This group consists of four G-protein coupled receptors (PAR-1 to -4) that show a unique activation characteristic [11]. In the presence of a protease, a portion of the PAR extracellular N-terminus is cleaved which then exposes a new N-terminus sequence that acts as a tethered ligand which then binds to the second extracellular loop of the receptor [12]. This conformational change to the receptor leads to intracellular signalling that will vary depending on the receptor subtype, the cleaving protease, and the downstream pathway that becomes activated. In addition to receptor activation, some proteases can disarm or inactivate the PAR receptor by cleaving the N-terminus at an adjacent site [13]. More recently, a biased signalling of PARs has been extensively described, indicating the complexity of these receptors [14–16]. PARs have been identified on the nerve terminals of nociceptors in multiple tissues suggesting that this receptor family is involved in pain control. Co-localization and pharmacological interaction between PAR-2 and pro-algesic transient receptor potential (TRP) channels in the pancreas [17], bladder [18], and oral mucosa [19] indicate that there is functional coupling between PARs and other known pain-modulating receptors. Proteases released from immunocytes, endothelial cells, or are part of the coagulation cascade are known to cleave neuronal PARs and modulate pain neurotransmission [12]. Other studies on the skin, gut, and airways have shown that PAR activation can lead to the secondary release or pro-algesic neuropeptides such as substance P and calcitonin gene-related peptides [20–22]. These multiple lines of evidence assert that PARs are an attractive target for the management of miscellaneous chronic pain conditions.

Table 1. Known cleaving proteases and synthetic activating peptides for each of the PARs and their role in arthritis.

Synthetic Activating PAR Activating Proteases Effect in Joints Peptides Thrombin SFLLRN-NH2 Chondroprotection [23] Granzyme A TFLLRN-NH2 Bone repair [24] Plasmin Pannus formation [25] Activating protein C Anti-allodynic via an opioid mechanism [26] Trypsin Factor Xa PAR-1 Kallikrein- 4, 5, 6, 14 MMP-1 Cathepsin G Proatherocytin Pen C 13 Chymase Trypsin SLIGRL-NH2 Cartilage degeneration [27] Synovial hyperaemia and increased Mast cell tryptase FLIGRL-NH2 leukocyte trafﬁcking [28] TRPV -dependent afferent sensitization and Factor Xa: Factor VIIa 1 pain [29,30] PAR-2 Acrosin Matriptase Serine 11D Trypsin Granzyme A Kallikrein-2, 4, 5, 6, 14 PAR-3 Thrombin N/A N/A Int. J. Mol. Sci. 2021, 22, 9352 3 of 16

Table 1. Cont.

Synthetic Activating PAR Activating Proteases Effect in Joints Peptides Thrombin AYPGKF-NH2 Joint damage [31] Trypsin GYPGKF-NH2 Joint hyperaemia and oedema [32] Afferent sensitization and pain via mast cell Cathepsin G degranulation and bradykinin activation [32] PAR-4 Trypsin IV Mannin-binding SP-1 Plasmin Factor Xa Kallikrein-1, 14 C4a

This review will outline the function of PARs and give an overview of the latest studies, implicating their role in joint diseases including RA and OA.

2. PAR-1 2.1. Receptor Pharmacology PAR-1 receptors are mainly activated by thrombin, but other mediators of the coagulation cascade can also cleave it. PAR-1 is ubiquitously found in endothelial cells, platelets, lungs, GI tract, immune cells, neurones and brain [11]. Following cleavage of PAR-1, there is activation of the G protein subunits G12/13,Gi, and Gq (Figure1A) [ 33–36]. Signalling via G12/13 leads stimulation of Rho guanine nucleotide exchange factor (RhoGEF), which in turn activates GTPase RhoA and is involved in cytoskeleton reorganization. Activation of Gi causes inhibition of adenylyl cyclase activity and hence a reduction in cyclic adenosine monophosphate (cAMP) production. Finally, Gq activation initiates the intracellular cascade starting with phospholipase-Cβ (CPLCβ) hydrolyzing phosphatidylinositol 4,5- bisphosphate (PIP2) to produce inositol triphosphate (IP3) and diacylglycerol (DAG). This leads to Ca2+ mobilization and an increase in the activity of protein kinase C (PKC) and other intracellular signalling enzymes. Different subunits of PKC facilitate the variety of downstream responses after PAR-1 activation [37–40]. A peculiar characteristic of PARs is that because its agonist is its own N-terminal sequence, there is no dissociation of the agonist from the receptor, so desensitization and termination mechanisms have to be tightly regulated. PAR-1 receptors are phosphorylated by G-protein coupled receptor kinases (GRKs) 3 or 5 and desensitized by β-arrestin binding, speciﬁcally β-arrestin 1, allowing the uncoupling of the G-proteins [12]. In contrast to other GPCRs, the β-arrestin associated with PAR-1 participates only in desensitization of the receptor, but not internalization [41]. Receptor internalization is primarily regulated by clathrin and dynamin activity. Adaptor protein complex 2 (AP-2) is essential for PAR-1 internalization, while lysosomal sorting and receptor recycling are mediated by the R4 subfamily of regulators of G protein signalling (RGS), viz. Rab11A and Rab11B [42–45]. Curiously, there is also an agonist-dependant response where activated protein C (APC) induces PAR-1 phosphorylation that does not result in effective internalization and the receptor accumulates in the cellular membrane [46,47]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 17

(PKC) and other intracellular signalling enzymes. Different subunits of PKC facilitate the variety of downstream responses after PAR-1 activation [37–40]. A peculiar characteristic of PARs is that because its agonist is its own N-terminal sequence, there is no dissociation of the agonist from the receptor, so desensitization and termination mechanisms have to be tightly regulated. PAR-1 receptors are phosphorylated by G-protein coupled receptor kinases (GRKs) 3 or 5 and desensitized by β-arrestin binding, specifically β-arrestin 1, allowing the uncoupling of the G-proteins [12]. In contrast to other GPCRs, the β-arrestin associated with PAR-1 participates only in desensitization of the receptor, but not internalization [41]. Receptor internalization is primarily regulated by clathrin and dynamin activity. Adaptor protein complex 2 (AP-2) is essential for PAR-1 internalization, while lysosomal sorting and receptor recycling are mediated by the R4 subfamily of regulators of G protein signalling (RGS), viz. Rab11A and Rab11B [42– 45]. Curiously, there is also an agonist-dependant response where activated protein C

Int. J. Mol. Sci. 2021, 22, 9352 (APC) induces PAR-1 phosphorylation that does not result in effective internalization4 ofand 16 the receptor accumulates in the cellular membrane [46,47].

Figure 1. Downstream signalling pathways for PAR-1 (A), PAR-2 (B), and PAR-4 (C). Rho guanine nucleotide exchange factor (RhoGEF); GTPase RhoA (RhoA); phospholipase-Cβ (CPLCβ); phosphatidylinositol 4,5-bisphosphate (PIP2); inositol

triphosphate (IP3); diacylglycerol (DAG); protein kinase C (PKC); cyclic adenosine monophosphate (cAMP). Figure 1. Downstream signalling pathways for PAR-1 (A), PAR-2 (B), and PAR-4 (C). Rho guanine nucleotide exchange factor (RhoGEF); GTPase RhoA2.2. PAR-1(RhoA); and phospholipase Joints -Cβ (CPLCβ); phosphatidylinositol 4,5-bisphosphate (PIP2); inositol triphosphate (IP3); diacylglycerol (DAG); protein kinase C (PKC); cyclic adenosine monophosphate (cAMP). Within joints, PAR-1 has been located on fibroblasts, myoblasts, osteoblasts, chondrocytes,2.2. PAR-1 and and synoviocytesJoints of RA and OA patients [48–50]. In joint cartilaginous tissue, thrombin stimulates migration and proliferation of chondrocytes in a PAR-1-dependent Within joints, PAR-1 has been located on fibroblasts, myoblasts, osteoblasts, chon- manner [23]. Under normal circumstances, migration of chondrocytes can help with car- drocytes, and synoviocytes of RA and OA patients [48–50]. In joint cartilaginous tissue, tilage homeostasis and tissue healing; however, in joint diseases such as OA and RA, thrombin stimulates migration and proliferation of chondrocytes in a PAR-1-dependent thrombin activation of those cells can initiate cartilage degradation [51], contributing to manner [23]. Under normal circumstances, migration of chondrocytes can help with car- arthritis progression. Metalloproteinases, such as MMP13, are elevated in OA and RA joints tilage homeostasis and tissue healing; however, in joint diseases such as OA and RA, and their catabolic properties lead to degradation of type II collagen through a PAR-1- and thrombin activation of those cells can initiate cartilage degradation [51], contributing to PAR-3-dependent pathway [52]. The same increase in expression of MMPs is observed with APC activity, but this seems to be through a mechanism other than PAR-1 activation [53]. In contrast, a protective role of PAR-1 in regulating osteoclast formation has also been reported. Kanno and collaborators showed that PAR-1 engagement can initiate a downstream response, culminating in phosphorylation of adenosine monophosphate kinase (AMPK) and inhibition of NF-kB signalling, resulting in reduced osteoclastogenesis. The authors showed an increase in urokinase plasminogen activating factor production following LPS treatment, and it is believed that subsequent plasmin production activated PAR-1 and reduced osteoclast formation [40]. These data were supported by another study showing that osteoclastogenesis was enhanced in PAR-1 knockout mice treated with TNF- alpha, indicating that PAR-1 acts as an inhibitor of osteoclast maturation in inflammatory joint diseases [54]. In a model of tibial damage, bone repair was found to be inhibited in PAR-1 knockout mice while thrombin improved bone marrow stromal cell proliferation in a PAR-1-dependent manner [24,55]. Thrombin is also able to inhibit osteoblast apoptosis, but in these cells this occurrence seems to be independent of PAR-1 activation [56]. In RA, synovial fibroblasts proliferate and form a hyperplastic and invasive tissue called pannus. Through PAR-1 cleavage, thrombin upregulates the expression of RANTES which then induces NF-kB and IL-6 expression, leading ultimately to synovial fibroblast Int. J. Mol. Sci. 2021, 22, 9352 5 of 16

differentiation and pannus formation [25,37,57,58]. In contrast, a protective role for PAR-1 in the joint has also been described. When treated with either thrombin, PAR-1-AP, or PAR-3-AP, synovial fibroblasts taken from OA patients have been shown to release heme- oxygenase-1 which is a protein with chondroprotective properties [38,59]. Thus, PAR-1 may be destructive or protective in the synovium depending upon whether the tissue is taken from a joint that has RA or OA, respectively. The role of PAR-1 in joints has also been tested pre-clinically using animal models of arthritis. In the antigen-induced arthritis (AIA) model, it was observed that treatment with the PAR-1 antagonist hirudin led to a reduction in joint diameter, pannus formation, cartilage damage, fibrin deposition, and release of IL-1β [60]. Similar results were found using the collagen-induced arthritis (CIA) model, where PEG-hirudin decreased all the parameters cited above in addition to decreasing IL-12 expression and disease incidence and severity [61]. Repeating the AIA model in PAR-1-knockout mice revealed that arthritic animals had less cartilage and bone damage, diminished overall disease severity, reduced IL-1β, IL-6 and MMP13 levels in the synovium, and consequently less synovitis [62]. Again, it appears that PAR-1 contributes to the severity and pathophysiology of inflammatory joint disease. In other studies, a link between PAR-1 and psoriatic arthritis has been observed. A transgenic mouse model that overexpresses kallikrein-related peptidase-6 (KLK6+) in keratinocytes, psoriatic arthritis markers such as IL-17A, IL-23 and IL-6 were all elevated [63]. Moreover, overexpression of KLK6+ caused increased expression of genes and proteins associated with psoriatic arthritis as well as pathological changes to the joint such as bone damage, synovitis, and impaired mobility. The PAR-1 antagonist vorapaxar inhibited the release of pro-inflammatory mediators from cultured keratinocytes, while PAR-1−/− mice that overexpress KLK6+ did not show the same level of psoriatic arthritis [63]. These results indicate that sustained activation of PAR-1 by KLK6 plays an important role in the development of psoriatic arthritis. With respect to pain, PAR-1 has divergent effects. The expression of PAR-1 in DRGs and its co-expression with TRPV1, SP and CGRP suggest a pro-nociceptive role for the receptor [64]. Thrombin injected into the dorsal horn of the spinal cord promoted the release of PGE2, leading to a decrease in mechanical withdrawal threshold [65], and an increase in glial cell expression [66]. Conversely, intraplantar injections of a synthetic PAR-1-AP was able to increase the withdrawal threshold in naïve animals without causing neuronal damage nor inflammation [67,68]. Following induction of inflammation with carrageenan, local injection of a PAR-1-AP was able to improve the hindpaw withdrawal threshold without affecting inflammation [67]. This analgesic effect of PAR-1 appears to involve the endogenous opioid system since naloxone was able to block PAR-1 responses [26]. The source of the opioids in inflamed tissues was found to be keratinocytes, as demonstrated by a rise in proenkephalin production by these cells. On balance, PAR-1 appears to be predominantly protective in inflammatory joint disease but may be involved in degenerative activity in OA. Further studies are needed to affirm the role of PAR-1 in different joint conditions as well as determining its possible contribution to joint pain.

3. PAR-2 3.1. Receptor Pharmacology PAR-2 is currently the most extensively characterized of all of the PARs. PAR-2 is the only PAR that does not have thrombin as the main endogenous activator. Rather, PAR-2 is predominantly cleaved by trypsin and tryptase, while other proteases such as neutrophil elastase and proteinase-3 are also known PAR-2 activators [14,15]. PAR-2 is highly expressed in epithelial cells of many systems such as lungs, liver, skin, blood vessels and the GI tract [69–73]. Expression of PAR-2 was also seen in mast cells [74], endothelial cells [75], different types of synoviocytes [76–79], joint sensory nerve ﬁbres [29,30] and dorsal root ganglia [80], showing the importance of this receptor in many systems. Int. J. Mol. Sci. 2021, 22, 9352 6 of 16

Similar to PAR-1, PAR-2 signalling involves Gq,G12/13 and Gi, although it is primarily mediated by Gq stimulating the PLCβ-IP3-DAG-PKC pathway (Figure1B) [ 43,81]. In some cells, activation of PAR-2 induces arachidonic acid release and fast production of prostaglandins [73], suggesting the involvement of phospholipase A2 and cyclooxygenase-1. Other cells show the involvement of mitogen-activated protein kinase (MAPK) and ERK1/2 pathways after stimulation of PAR-2, as well as a non-G-protein-dependent pathway mediated by β-arrestin [43,82,83]. Biased signalling via the PLC-IP3-DAG-PKC pathway leads to Ca2+ mobilization from intracellular stores, while the MAPK pathway leads to signalling that is Ca2+-independent. The desensitization and termination of the PAR-2 signal is slightly different from PAR-1. Instead of using GRKs, PAR-2 phosphorylation is accomplished by PKC [81], which increases its afﬁnity for clatharin, dynamin and β-arrestin 1 and 2, thereby facilitating the uncoupling of the receptor and its eventual internalization [84–86]. The internalization of PAR-2 involves β-arrestin with GTPases being important for endosome formation (Rab5a) and intracellular trafﬁcking (Rab11a) [35,87].

3.2. PAR-2 and Joints PAR-2 is widely expressed in multiple different tissues in the joint and is therefore likely to play a role in joint diseases such as OA and RA. In vitro studies showed that PAR-2 is expressed in osteoblasts and its activation induces calcium signalling and inhibition of osteoclast differentiation [76,88]. Compared to wild type animals, PAR-2−/− mice have a lower expression of osteoblasts and osteoclasts, suggesting that PAR-2 is involved in the regulation of skeletal growth and bone repair [89]. Conversely, PAR-2 activation of osteoblasts from OA donors led to an increase in MMP1, MMP9, IL-6 and RANKL levels, indicating a bone resorptive effect in diseased joints [90]. Chondrocytes collected from OA patients show heightened levels of PAR-2 and exposure of these cells to inflammatory cytokines increased the expression of the receptor [77,79]. In contrast, TGF-β caused a downregulation of PAR-2 expression in OA chondrocytes but not in healthy cells [77,91], implying that various inflammatory mediators may differentially regulate the expression of PAR-2 in chondrocytes during joint disease. Matriptase is a serine protease that is rich in OA cartilage where it was shown to cause collagenolysis through PAR-2 activation [27]. Huang and colleagues (2019) used an antagonist of PAR-2 in both in vitro and in vivo models to elucidate the activity of PAR-2 in chondrocytes during OA as a possible treatment for the condition. They observed that AZ3451 inhibited PAR-2 expression and many of its downstream signalling pathways in vitro, reducing the expression of pro-inflammatory cytokines, collagen type II, and catabolic genes. In in vivo experiments, the antagonist inhibited cartilage destruction, decreased MMP13 levels and inhibited chondrocyte apoptosis [92]. Thus, PAR-2 may be a useful target to curtail cartilage damage during OA progression. In synovial fibroblasts from human RA samples, the presence of PAR-2 receptors was detected, and stimulation of the receptor induced Ca2+ mobilization. Unlike chondrocytes, exposure of the synoviocytes to IL-1β and TNF-α did not increase the expression of PAR-2 in those cells [93]. In RA samples, mast cell tryptase activated PAR-2 on synovial fibroblasts inhibiting apoptosis via a rho kinase mechanism [94]. Both mast cell tryptase and a PAR-2-AP were able to induce synovial fibroblast proliferation in cells cultured from RA and OA patients with RA samples showing the greatest effect [95]. Analysis of venous blood samples from RA patients discovered that PAR-2 receptors were highly expressed on monocytes and T-cells, and generation of IL-6 by monocytes after PAR-2 activation was more prominent in blood from RA patients. PAR-2 expression in blood samples taken from OA patients revealed that the receptor levels were close to what was seen in control samples [96]. However, in OA patients with concomitant synovitis, PAR-2 receptor expression was elevated in the synovium and on macrophages, lymphocytes, and fibroblasts [97]. Measured levels of PAR-2 correlated with the severity of the synovitis, suggesting that PAR-2 is relevant in joint disease where there is ongoing inflammation. In other experiments, blocking PAR-2 reduced pannus invasion and proliferation, decreased Int. J. Mol. Sci. 2021, 22, 9352 7 of 16

the release of IL-17, IL-1β, and TNF-α, and ameliorated signs of OA via regulation of the MAPK/NF-kB pathway [50,98,99]. Direct injection of a PAR-2-AP into the joint was able to cause long lasting joint swelling, hyperemia, and synovial vasodilation in wild type mice, but not in their lit- termates lacking PAR-2 [100]. Degranulation of joint connective tissue mast cells with compound 48/80 produced an inflammatory response in wild type mice but not PAR-2 knockout animals, highlighting that mast cells are involved in the PAR-2 inflammation pathway [78]. Nociceptive behavior after an intraarticular injection of a PAR-2-AP was first observed by Helyes et al. (2010), where they were able to show that SLIGRL-NH2 could decrease paw withdrawal threshold and increase joint incapacitance [29]. This pro-algesic effect of PAR-2 activation was TRPV1-dependent since the selective TRPV1 antagonist SB366791 blocked this response. Articular injection of the more potent PAR-2-AP FLIGRL- NH2 induced spontaneous activity and increased firing of knee joint primary afferents in rats that was blocked by both TRPV1 and NK1 receptor antagonists [30]. In addition to a peripheral site of action for PAR-2, intra-thecal injection of FLIGRL-NH2 heightened mechanonociception in naïve rats [101]. Furthermore, spinal administration of the PAR-2 antagonist GB83 reduced secondary allodynia in models of chronic arthritis, but not in acute synovitis [101]. Cleaving serine proteases have also been shown to induce joint inflammation and pain in a PAR-2-dependent manner. For example, local administration of neutrophil elastase caused an increase in synovial blood perfusion, leukocyte trafficking, and nociceptive behaviour in wild type mice but not PAR-2−/− [28]. Mast cell tryptase delivered into the knee caused synovial hyperaemia, oedema, and pain behaviour, which was reduced by −/− chemical ablation of TRPV1 expressing neurones or in TRPV1 animals [102]. Similarly, another PAR-2 cleaving enzyme, matriptase, can increase synovial perfusion in wild type mice but not PAR-2−/− [27]. In addition to activating proteases, some enzymes can silence PAR activity by cleaving downstream of the canonical cleavage site. Calpain I, for example, disarms PAR-2 in joints, leading to a reduction in pain behaviour [103]. Experiments investigating a role for PAR-2 in chronic inflammatory joint disease found that in wild type mice, intra-articular injection of Freund’s complete adjuvant produced inflammatory cellular infiltration, synovial hyperplasia, and cartilage damage; however, none of these processes occurred in PAR-2−/− mice, confirming that PAR-2 plays a role in joint inflammation [100]. PAR-2 has also been found to be involved in acute joint inflammation models where either deletion of the PAR-2 gene or inhibition of the receptor by RNA silencing reduced evidence of synovitis [100,104]. PAR-2−/− mice were tested for their ability to develop RA in antigen-induced models and there was impaired development of the disease, indicating that PAR-2 is involved in the adaptative immune response to inflammatory joint disease [105,106]. PAR-2 has also been investigated in animal models of OA. Following joint destabiliza- tion, OA gradually developed in wild type but not PAR-2−/− mice [107–110], indicating that PAR-2 is also involved in OA development. More recently, using chemical induction of OA, Muley and colleagues found that neutrophil elastase activity was increased in the joint of rats after induction of OA [111]. Pharmacological inhibition of this serine protease with alpha-antitrypsin or treatment with a PAR-2 antagonist blocked the initial inflammatory response to neutrophil elastase and reduced pain and peripheral neuropathy [111]. In a rodent model of chronic joint inflammation, alpha-antitrypsin reduced joint pain and leukocyte trafficking and exerted a chondroprotective effect [112]. It is clear that PAR-2 participates in the development of both RA and OA; therefore, inhibition of this receptor and its pathways presents a promising approach for the treatment of chronic joint diseases and their symptoms. Int. J. Mol. Sci. 2021, 22, 9352 8 of 16

4. PAR-3 4.1. Receptor Pharmacology The observation that platelets from PAR-1-knockout mice could still respond to thrombin led to the identiﬁcation, cloning and characterization of PAR-3 [113,114]. A great limitation to the study of PAR-3 is the fact that synthetic activating peptides based on the receptor tethered ligand sequence do not activate PAR-3 [114]. It was later shown that these so-called PAR-3-Aps actually bound to both PAR-1 and PAR-2 instead [115]. As such, the signalling pathways of PAR-3 are still obscure, and it seems to play a more important role as a cofactor for other PARs as a regulator of their activity through heterodimeriza- tion [116–118].

4.2. PAR-3 and Joints PAR-3 is co-expressed with PAR-1 on human chondrocytes but no functional activity of PAR-3 in those cells has been observed [23]. In synovial fibroblasts from RA samples, PAR-3 is co-expressed with PAR-1 and can cause a small Ca2+ influx itself, but not enough to alter cell activity [58]. In contrast, thrombin and a PAR-3-AP can stimulate production of heme oxygenase-1 in synovial fibroblasts from OA patients, and this effect can be inhibited by siRNA blockade of PAR-3 [38]. However, since the PAR-3-AP is not selective and PAR-3 is co-expressed with PAR-1 in synovial fibroblasts, it is feasible that these responses are actually a result of PAR-1 activation and PAR-3 is acting here as a cofactor to regulate PAR-1 activity. The main hindrance to PAR-3 biology has been the lack of selective PAR-3 agonists and antagonists. However, the recent discovery of a PAR-3 lipid tethered binding peptide (C660) may reveal a possible role for PAR-3 in arthritic conditions [119].

5. PAR-4 5.1. Receptor Pharmacology PAR-4 was identified and cloned after the observation that platelets lacking PAR-1 and PAR-3 still responded to thrombin [120,121]. PAR-4 can be activated by thrombin, cathepsin G, tissue kallikreins, trypsin, and coagulation factors, via both Gq and G12/13 signalling pathways (Figure1C). G 12/13 follows the RhoGEF, GTPase RhoA, and downstream signalling pathways in order to alter the conformation of platelets and initiate aggregation. Engagement of the Gq second messenger system leads to the activation of 2+ PLCβ, production of IP3 and DAG, leading to Ca mobilization and enhancement of PKC activity [122,123]. It has been shown that PAR-4 signalling and termination occurs at a much slower pace than PAR-1. First PAR-1 is activated and promotes an acute Ca2+ increase followed by a prolonged and sustained Ca2+ signal elicited by PAR-4 activation [124,125]. One of the reasons PAR-4 can sustain the signalling is based on the C-terminus being much shorter than other PARs and it having less sites for phosphorylation than are necessary for receptor desensitization and internalization [124]. Furthermore, PAR-4 internalization pro- ceeds independently of β-arrestin but rather occurs via clatherin coating and endocytosis. Nevertheless, regulators of G protein signalling (RGS) eventually recognize the receptor and form complexes to terminate its signalling. Specifically, RGS2 interacts with Gq and RGS4 interacts with G12/13, inhibiting the downstream processes of these G proteins [126]. Upon internalization, PAR-4 is sorted between lysosomes for degradation or endosomes for recycling [122]. Interestingly, the recycling of PAR-4 is more efficient when it forms a dimer with PAR-2, resulting in a faster process of delivering PAR-4 to the cell membrane [127]. Dimerization with PAR-1 or PAR-3 can also influence PAR-4 activity by potentiating or modulating its signalling properties [128,129].

5.2. PAR-4 and Joints PAR-4 receptors are expressed throughout the knee joint, as seen from positive im- munohistochemical staining in articular cartilage, subchondral bone, menisci, synovium, mast cells, and chondrocytes. [31,32,130,131]. Intra-articular injection of the neuronal tracer ﬂuorogold revealed that PAR-4 is expressed on joint primary afferents, suggesting that Int. J. Mol. Sci. 2021, 22, 9352 9 of 16

the receptor has the potential to modulate nociceptor firing. In a model of arthritis, the level of PAR-4 in the joint positively correlated with monocyte infiltration and synovial hypertrophy, indicating that PAR-4 plays a role in joint inflammation [97]. In contrast to the anti-inflammatory and analgesic effect of PAR-4 activation in skin and the gut [132], intraarticular injection of a PAR-4-AP caused a long-lasting increase in synovial blood flow, oedema formation, and an increase in mechanical and thermal pain [32]. Recently, it has been found that PAR-4 is involved in joint damage associated with articular bleeds. The accumulation of blood in a joint, for example, as a result of trauma, can lead to arthropathy, including synovitis and cartilage damage. Silencing of PAR-4 using siRNA reduced synovitis scores and hyaline cartilage destruction in mice with an intra-articular bleed [31]. It was suggested that this pro-arthritic effect was in part mediated by increased plasmin levels cleaving PAR-4 in the joint. Electrophysiological recordings from joint primary afferents revealed an increase in nerve firing after close intra-arterial injection of a PAR-4-AP confirming a pro-nociceptive role for PAR-4 in joints. The inflammatory and nociceptive effects of PAR-4 activation could be blocked by a PAR-4 antagonist or the bradykinin B2 receptor antagonist HOE140 [130]. Conversely, the TRPV1 antagonist SB366791 did not influence PAR-4 responses. Pre- treatment of rats with a mast cell stabilizer also blocked mechanosensitization and pain implicating a mast cell-kinin mechanism for PAR-4 activity [131]. In other studies, silencing of PAR-4 with siRNA treatment reduced the severity of joint damage and plasmin activity in a model of blood-induced arthropathy [31]. Together, these data imply that inhibition of PAR-4 activity can be used to treat joint inflammation and pain.

6. Conclusions The family of PARs are richly expressed throughout joints and are associated with tissues associated with pain control, inflammation, and structural damage. On balance, PARs appear to be promoters of heightened inflammation and pain, while their contribution to joint destruction may be disease-dependent and requires further exploration (for summary, see Figure2). Growing evidence seems to indicate that PAR-1 and PAR-2 can cause innate and neurogenic inflammation in different arthritis models, while PAR-4 seems to require platelet–leukocyte interactions, mast cell degranulation, and involvement of the kallikrein-kinin systems. When it comes to pain transmission, PAR-1 imparts an analgesic effect, whereas PAR-2 and PAR-4 support a pro-nociceptive outcome. The role of PAR-3 in joints is still unclear and we await better tools and techniques to unravel the function of this receptor in arthritis pathogenesis and symptom development. The dual effects of serine proteases to induce joint tissue catabolism and signal pain and inflammation make them promising candidates to redress the destructive and painful features of arthritis. Int. J. Mol. Sci. 22 Int. J. Mol. Sci.2021 2021,, 22,, 9352 x FOR PEER REVIEW 1110 ofof 1617

FigureFigure 2. 2.Overview Overview ofof thethe rolerole ofof PARsPARs in in the the control control of of joint joint inﬂammation inflammation andand pain.pain. ActivationActivation ofof aa PARPAR isis achievedachieved byby cleavagecleavage ofof thethe receptorreceptor withwith aa proteolyticproteolytic enzymeenzyme toto revealreveal aa tetheredtethered ligandligand thatthat bindsbinds toto thethe secondsecond extracellularextracellular loop,loop, leadingleading to to G G protein protein coupling coupling and and signallingsignalling ((AA).). InIn joints,joints, PAR-2PAR-2 andand -4-4 tendtend toto bebe pro-inﬂammatorypro-inflammatory andand pro-nociceptive,pro-nociceptive, whereas PAR-1 has some protective properties (B). whereas PAR-1 has some protective properties (B).

Int. J. Mol. Sci. 2021, 22, 9352 11 of 16

Author Contributions: F.L. wrote the original draft, edited, and reviewed the final version of the manuscript. J.J.M. edited and reviewed the final version of the manuscript. Both authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Figures by M.S. O’Brien. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Woolf, A.; Pfleger, B. Burden of major muskuloskeletal conditions. Bull. World Health Organ. 2003, 81, 646–656. 2. van Delft, M.A.M.; Huizinga, T.W.J. An overview of autoantibodies in rheumatoid arthritis. J. Autoimmun. 2020, 110, 102392. [CrossRef] 3. Grässel, S.; Zaucke, F.; Madry, H. Osteoarthritis: Novel Molecular Mechanisms Increase Our Understanding of the Disease Pathology. J. Clin. Med. 2021, 10, 1938. [CrossRef] 4. Berenbaum, F. Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthr. Cartil. 2013, 21, 16–21. [CrossRef][PubMed] 5. Lee, D.M.; Weinblatt, M.E. Rheumatoid arthritis. Lancet 2001, 358, 903–911. [CrossRef] 6. Helmick, C.G.; Felson, D.T.; Lawrence, R.C.; Gabriel, S.; Hirsch, R.; Kwoh, C.K.; Liang, M.H.; Kremers, H.M.; Mayes, M.D.; Merkel, P.A.; et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States: Part I. Arthritis Rheum. 2007, 58, 15–25. [CrossRef] 7. Gibofsky, A. Overview of epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis. Am. J. Manag. Care 2012, 18, S295–S302. 8. Singh, J.A.; Saag, K.G., Jr.; Akl, E.A.; Bannuru, R.R.; Sullivan, M.C.; Vaysbrot, E.; McNaughton, C.; Osani, M.; Shmerling, R.H.; McAlindon, T.; et al. 2015 American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis. Arthritis Rheumatol. 2015, 68, 1–26. [CrossRef] 9. McAlindon, T.E.; Bannuru, R.R.; Sullivan, M.C.; Arden, N.K.; Berenbaum, F.; Bierma-Zeinstra, S.M.; Hawker, G.A.; Henrotin, Y.; Hunter, D.J.; Kawaguchi, H.; et al. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthr. Cartil. 2014, 22, 363–388. [CrossRef] 10. Krustev, E.; Rioux, D.; McDougall, J.J. Mechanisms and Mediators That Drive Arthritis Pain. Curr. Osteoporos. Rep. 2015, 13, 216–224. [CrossRef][PubMed] 11. Hollenberg, M.D. Protease-mediated signalling: New paradigms for cell regulation and drug development. Trends Pharmacol. Sci. 1996, 17, 3–6. [CrossRef] 12. McDougall, J.J.; Muley, M.M. The Role of Proteases in Pain. In Pain Control; Schaible, H.-G., Ed.; Springer: Berlin/Heidelberg, Germany, 2015; pp. 239–260. ISBN 978-3-662-46450-2. 13. Ramachandran, R.; Noorbakhsh, F.; DeFea, K.; Hollenberg, M.D. Targeting proteinase-activated receptors: Therapeutic potential and challenges. Nat. Rev. Drug Discov. 2012, 11, 69–86. [CrossRef] 14. Zhao, P.; Metcalf, M.; Bunnett, N.W. Biased Signaling of Protease-Activated Receptors. Front. Endocrinol. 2014, 5, 67. [CrossRef] 15. Hollenberg, M.D.; Mihara, K.; Polley, D.; Suen, J.Y.; Han, A.; Fairlie, D.P.; Ramachandran, R. Biased signalling and proteinase- activated receptors (PARs): Targeting inflammatory disease. Br. J. Pharmacol. 2013, 171, 1180–1194. [CrossRef][PubMed] 16. Willis-Fox, O.; Preston, R.J.S. Molecular basis of protease-activated receptor 1 signaling diversity. J. Thromb. Haemost. 2019, 18, 6–16. [CrossRef] 17. Hoogerwerf, W.A.; Zou, L.; Shenoy, M.; Sun, D.; Micci, M.A.; Lee-Hellmich, H.; Xiao, S.Y.; Winston, J.H.; Pasricha, P.J. The Proteinase-Activated Receptor 2 Is Involved in Nociception. J. Neurosci. 2001, 21, 9036–9042. [CrossRef] 18. Dattilio, A.; Vizzard, M.A. Up-regulation of protease activated receptors in bladder after cyclophosphamide induced cystitis and colocalization with capsaicin receptor (vr1) in bladder nerve fibers. J. Urol. 2005, 173, 635–639. [CrossRef] 19. Ito, M.; Ono, K.; Hitomi, S.; Nodai, T.; Sago, T.; Yamaguchi, K.; Harano, N.; Gunjigake, K.; Hosokawa, R.; Kawamoto, T.; et al. Prostanoid-dependent spontaneous pain and PAR2-dependent mechanical allodynia following oral mucosal trauma. Mol. Pain 2017, 13, 1–17. [CrossRef] 20. Vergnolle, N.; Bunnett, N.W.; Sharkey, K.; Brussee, V.; Compton, S.J.; Grady, E.F.; Cirino, G.; Gerard, N.; Basbaum, A.I.; Andrade-Gordon, P.; et al. Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway. Nat. Med. 2001, 7, 821–826. [CrossRef] 21. Grant, A.D.; Cottrell, G.S.; Amadesi, S.; Trevisani, M.; Nicoletti, P.; Materazzi, S.; Altier, C.; Cenac, N.; Zamponi, G.W.; Bautista-Cruz, F.; et al. Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J. Physiol. 2007, 578, 715–733. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 9352 12 of 16

22. Obreja, O.; Rukwied, R.; Steinhoff, M.; Schmelz, M. Neurogenic components of trypsin- and thrombin-induced inflammation in rat skin, in vivo. Exp. Dermatol. 2006, 15, 58–65. [CrossRef] 23. Kirilak, Y.; Pavlos, N.J.; Willers, C.R.; Han, R.; Feng, H.; Xu, J.; Asokananthan, N.; Stewart, G.A.; Henry, P.; Wood, D.; et al. Fibrin sealant promotes migration and proliferation of human articular chondrocytes: Possible involvement of thrombin and protease-activated receptors. Int. J. Mol. Med. 2006, 17, 551–558. [CrossRef] 24. Song, S.J.; Pagel, C.N.; Campbell, T.M.; Pike, R.N.; Mackie, E.J. The Role of Protease-Activated Receptor-1 in Bone Healing. Am. J. Pathol. 2005, 166, 857–868. [CrossRef] 25. Hirano, F.; Kobayashi, A.; Hirano, Y.; Nomura, Y.; Fukawa, E.; Makino, I. Thrombin-induced expression of RANTES mRNA through protease activated receptor-1 in human synovial fibroblasts. Ann. Rheum. Dis. 2002, 61, 834–837. [CrossRef] 26. Martin, L.; Augé, C.; Boué, J.; Buresi, M.C.; Chapman, K.; Asfaha, S.; Andrade-Gordon, P.; Steinhoff, M.; Cenac, N.; Dietrich, G.; et al. Thrombin receptor: An endogenous inhibitor of inflammatory pain, activating opioid pathways. Pain 2009, 146, 121–129. [CrossRef] 27. Milner, J.M.; Patel, A.; Davidson, R.K.; Swingler, T.E.; Désilets, A.; Young, D.A.; Kelso, E.B.; Donell, S.T.; Cawston, T.E.; Clark, I.M.; et al. Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis. Arthritis Rheum. 2010, 62, 1955–1966. [CrossRef][PubMed] 28. Muley, M.M.; Reid, A.R.; Botz, B.; Bölcskei, K.; Helyes, Z.; McDougall, J.J. Neutrophil elastase induces inflammation and pain in mouse knee joints via activation of proteinase-activated receptor-2. Br. J. Pharmacol. 2015, 173, 766–777. [CrossRef][PubMed] 29. Helyes, Z.; Sándor, K.; Borbély, É.; Tékus, V.; Pintér, E.; Elekes, K.; Tóth, D.M.; Szolcsányi, J.; McDougall, J.J. Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception. Eur. J. Pain 2010, 14, 351–358. [CrossRef] 30. Russell, F.A.; Schuelert, N.; Veldhoen, V.E.; Hollenberg, M.D.; McDougall, J.J. Activation of PAR2receptors sensitizes primary afferents and causes leukocyte rolling and adherence in the rat knee joint. Br. J. Pharmacol. 2012, 167, 1665–1678. [CrossRef] [PubMed] 31. Nieuwenhuizen, L.; Schutgens, R.E.G.; Coeleveld, K.; Mastbergen, S.C.; Schiffelers, R.M.; Roosendaal, G.; Biesma, D.H.; Lafeber, F.P.J.G. Silencing of protease-activated receptors attenuates synovitis and cartilage damage following a joint bleed in haemophilic mice. Haemophilia 2015, 22, 152–159. [CrossRef] 32. McDougall, J.J.; Zhang, C.; Cellars, L.; Joubert, E.; Dixon, C.M.; Vergnolle, N. Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice. Arthritis Rheum. 2009, 60, 728–737. [CrossRef][PubMed] 33. Hung, D.T.; Wong, Y.H.; Vu, T.K.H.; Coughlin, S.R. The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase. J. Biol. Chem. 1992, 267, 20831–20834. [CrossRef] 34. Benka, M.L.; Lee, M.; Wang, G.-R.; Buckman, S.; Burlacu, A.; Cole, L.; DePina, A.; Dias, P.; Granger, A.; Grant, B.; et al. The thrombin receptor in human platelets is coupled to a GTP binding protein of the Gαqfamily. FEBS Lett. 1995, 363, 49–52. [CrossRef] 35. Déry, O.; Corvera, C.U.; Steinhoff, M.; Bunnett, N.W. Proteinase-activated receptors: Novel mechanisms of signaling by serine proteases. Am. J. Physiol. Physiol. 1998, 274, C1429–C1452. [CrossRef] 36. Ossovskaya, V.S.; Bunnett, N.W. Protease-Activated Receptors: Contribution to Physiology and Disease. Physiol. Rev. 2004, 84, 579–621. [CrossRef] 37. Chiu, Y.-C.; Fong, Y.-C.; Lai, C.-H.; Hung, C.-H.; Hsu, H.-C.; Lee, T.-S.; Yang, R.-S.; Fu, W.-M.; Tang, C.-H. Thrombin-induced IL-6 production in human synovial fibroblasts is mediated by PAR1, phospholipase C, protein kinase Cα, c-Src, NF-kappaB and p300 pathway. Mol. Immunol. 2008, 45, 1587–1599. [CrossRef][PubMed] 38. Liu, J.-F.; Hou, S.-M.; Tsai, C.-H.; Huang, C.-Y.; Yang, W.-H.; Tang, C.-H. Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways. Arthritis Res. Ther. 2012, 14, R91. [CrossRef] 39. Tao, K.-M.; Tao, Y.; Chen, C.-Y.; Yang, L.-Q.; Lu, Z.-J.; Sun, Y.-M.; Huang, S.-D.; Yu, W.-F. Proteinase-activated Receptor 1 Contributed to Up-regulation of Enkephalin in Keratinocytes of Patients with Obstructive Jaundice. Anesthesiology 2014, 121, 127–139. [CrossRef] 40. Kanno, Y.; Ishisaki, A.; Kawashita, E.; Kuretake, H.; Ikeda, K.; Matsuo, O. uPA Attenuated LPS-induced Inflammatory Osteoclas- togenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis. Int. J. Biol. Sci. 2016, 12, 63–71. [CrossRef] 41. Paing, M.M.; Stutts, A.B.; Kohout, T.A.; Lefkowitz, R.J.; Trejo, J. β-Arrestins Regulate Protease-activated Receptor-1 Desensitization but Not Internalization or Down-regulation. J. Biol. Chem. 2002, 277, 1292–1300. [CrossRef] 42. Paing, M.M.; Johnston, C.A.; Siderovski, D.P.; Trejo, J. Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization. Mol. Cell. Biol. 2006, 26, 3231–3242. [CrossRef] 43. Adams, M.N.; Ramachandran, R.; Yau, M.-K.; Suen, J.Y.; Fairlie, D.P.; Hollenberg, M.D.; Hooper, J.D. Structure, function and pathophysiology of protease activated receptors. Pharmacol. Ther. 2011, 130, 248–282. [CrossRef][PubMed] 44. Chen, B.; Siderovski, D.P.; Neubig, R.R.; Lawson, M.A.; Trejo, J. Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins. J. Biol. Chem. 2014, 289, 1580–1591. [CrossRef] 45. Grimsey, N.J.; Coronel, L.J.; Cordova, I.C.; Trejo, J. Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins. J. Biol. Chem. 2016, 291, 2223–2236. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 9352 13 of 16

46. Schuepbach, R.A.; Feistritzer, C.; Brass, L.F.; Riewald, M. Activated protein C–cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin. Blood 2008, 111, 2667–2673. [CrossRef] 47. Russo, A.; Soh, U.J.K.; Paing, M.M.; Arora, P.; Trejo, J. Caveolae are required for protease-selective signaling by protease-activated receptor-1. Proc. Natl. Acad. Sci. USA 2009, 106, 6393–6397. [CrossRef][PubMed] 48. Morris, R.; Winyard, P.G.; Brass, L.F.; Blake, D.R.; Morris, C.J. Thrombin receptor expression in rheumatoid and osteoarthritic synovial tissue. Ann. Rheum. Dis. 1996, 55, 841–843. [CrossRef] 49. Shin, H.; Nakajima, T.; Kitajima, I.; Shigeta, K.; Abeyama, K.; Imamura, T.; Okano, T.; Kawahara, K.; Nakamura, T.; Maruyama, I. Thrombin Receptor-Mediated Synovial Proliferation in Patients with Rheumatoid Arthritis. Clin. Immunol. Immunopathol. 1995, 76, 225–233. [CrossRef] 50. Xue, M.; Chan, Y.-K.A.; Shen, K.; Dervish, S.; March, L.; Sambrook, P.N.; Jackson, C.J. Protease-activated receptor 2, rather than protease-activated receptor 1, contributes to the aggressive properties of synovial fibroblasts in rheumatoid arthritis. Arthritis Rheum. 2011, 64, 88–98. [CrossRef] 51. Furmaniak-Kazmierczak, E.; Cooke, T.D.; Manuel, R.; Scudamore, A.; Hoogendorn, H.; Giles, A.R.; Nesheim, M. Studies of thrombin-induced proteoglycan release in the degradation of human and bovine cartilage. J. Clin. Investig. 1994, 94, 472–480. [CrossRef] 52. Huang, C.-Y.; Lin, H.-J.; Chen, H.-S.; Cheng, S.-Y.; Hsu, H.-C.; Tang, C.-H. Thrombin Promotes Matrix Metalloproteinase-13 Expression through the PKCδ/c-Src/EGFR/PI3K/Akt/AP-1 Signaling Pathway in Human Chondrocytes. Mediat. Inflamm. 2013, 2013, 326041. [CrossRef] 53. Jackson, M.T.; Moradi, B.; Smith, M.M.; Jackson, C.J.; Little, C.B. Activation of Matrix Metalloproteinases 2, 9, and 13 by Activated Protein C in Human Osteoarthritic Cartilage Chondrocytes. Arthritis Rheumatol. 2014, 66, 1525–1536. [CrossRef] 54. Jastrzebski, S.; Kalinowski, J.; Mun, S.; Shin, B.; Adapala, N.S.; Jacome-Galarza, C.E.; Mirza, F.; Aguila, H.L.; Drissi, H.; Sanjay, A.; et al. Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism. J. Immunol. 2019, 203, 105–116. [CrossRef] 55. Song, S.J.; Pagel, C.N.; Pike, R.N.; Mackie, E.J. Studies on the receptors mediating responses of osteoblasts to thrombin. Int. J. Biochem. Cell Biol. 2005, 37, 206–213. [CrossRef] 56. Pagel, C.N.; de Niese, M.R.; Abraham, L.A.; Chinni, C.; Song, S.-J.; Pike, R.N.; Mackie, E.J. Inhibition of osteoblast apoptosis by thrombin. Bone 2003, 33, 733–743. [CrossRef] 57. Shin, H.; Kitajima, I.; Nakajima, T.; Shao, Q.; Tokioka, T.; Takasaki, I.; Hanyu, N.; Kubo, T.; Maruyama, I. Thrombin receptor mediated signals induce expressions of interleukin 6 and granulocyte colony stimulating factor via NF-kappa B activation in synovial fibroblasts. Ann. Rheum. Dis. 1999, 58, 55–60. [CrossRef][PubMed] 58. Furuhashi, I.; Abe, K.; Sato, T.; Inoue, H. Thrombin-Stimulated Proliferation of Cultured Human Synovial Fibroblasts through Proteolytic Activation of Proteinase-Activated Receptor-1. J. Pharmacol. Sci. 2008, 108, 104–111. [CrossRef][PubMed] 59. Guillén, M.; Megías, J.; Gomar, F.; Alcaraz, M. Haem oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes. J. Pathol. 2007, 214, 515–522. [CrossRef] 60. Varisco, P.A.; Péclat, V.; Van Ness, K.; Bischof-Delaloye, A.; So, A.; Busso, N. Effect of thrombin inhibition on synovial inflammation in antigen induced arthritis. Ann. Rheum. Dis. 2000, 59, 781–787. [CrossRef] 61. Marty, I.; Péclat, V.; Kirdaite, G.; Salvi, R.; So, A.; Busso, N. Amelioration of collagen-induced arthritis by thrombin inhibition. J. Clin. Investig. 2001, 107, 631–640. [CrossRef][PubMed] 62. Yang, Y.H.; Hall, P.; Little, C.B.; Fosang, A.J.; Milenkovski, G.; Santos, L.; Xue, J.; Tipping, P.; Morand, E.F. Reduction of arthritis severity in protease-activated receptor-deficient mice. Arthritis Rheum. 2005, 52, 1325–1332. [CrossRef] 63. Billi, A.C.; Ludwig, J.E.; Fritz, Y.; Rozic, R.; Swindell, W.R.; Tsoi, L.C.; Gruszka, D.; Abdollahi-Roodsaz, S.; Xing, X.; Diaconu, D.; et al. KLK6 expression in skin induces PAR1-mediated psoriasiform dermatitis and inflammatory joint disease. J. Clin. Investig. 2020, 130, 3151–3157. [CrossRef] 64. Vellani, V.; Kinsey, A.M.; Prandini, M.; Hechtfischer, S.C.; Reeh, P.; Magherini, P.C.; Giacomoni, C.; McNaughton, P.A. Protease Activated Receptors 1 and 4 Sensitize TRPV1 in Nociceptive Neurones. Mol. Pain 2010, 6, 61. [CrossRef][PubMed] 65. Koetzner, L.; Gregory, J.A.; Yaksh, T.L. Intrathecal Protease-Activated Receptor Stimulation Produces Thermal Hyperal-gesia through Spinal Cyclooxygenase Activity. J. Pharmacol. Exp. Ther. 2004, 311, 356–363. [CrossRef][PubMed] 66. Smith, J.R.; Syre, P.P.; Oake, S.A.; Nicholson, K.J.; Weisshaar, C.L.; Cruz, K.; Bucki, R.; Baumann, B.C.; Janmey, P.A.; Winkelstein, B.A. Salmon and Human Thrombin Differentially Regulate Radicular Pain, Glial-Induced Inflammation and Spinal Neuronal Excitability through Protease-Activated Receptor-1. PLoS ONE 2013, 8, e80006. [CrossRef] 67. Asfaha, S.; Brussee, V.; Chapman, K.; Zochodne, D.W.; Vergnolle, N. Proteinase-activated receptor-1 agonists attenuate nociception in response to noxious stimuli. Br. J. Pharmacol. 2002, 135, 1101–1106. [CrossRef] 68. Kawabata, A.; Kawao, N.; Kuroda, R.; Tanaka, A.; Shimada, C. The PAR-1-activating peptide attenuates carrageenan-induced hyperalgesia in rats. Peptides 2002, 23, 1181–1183. [CrossRef] 69. Al-Ani, B.; Saifeddine, M.; Kawabata, A.; Renaux, B.; Mokashi, S.; Hollenberg, M.D. Proteinase-activated receptor 2 (PAR(2)): Development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands. J. Pharmacol. Exp. Ther. 1999, 290, 753–760. Int. J. Mol. Sci. 2021, 22, 9352 14 of 16

70. Santulli, R.J.; Derian, C.K.; Darrow, A.L.; Tomko, K.A.; Eckardt, A.J.; Seiberg, M.; Scarborough, R.M.; Andrade-Gordon, P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc. Natl. Acad. Sci. USA 1995, 92, 9151–9155. [CrossRef][PubMed] 71. Hwa, J.J.; Ghibaudi, L.; Williams, P.; Chintala, M.; Zhang, R.; Chatterjee, M.; Sybertz, E. Evidence for the Presence of a Proteinase- Activated Receptor Distinct From the Thrombin Receptor in Vascular Endothelial Cells. Circ. Res. 1996, 78, 581–588. [CrossRef] [PubMed] 72. Saifeddine, M.; Al-Ani, B.; Cheng, C.-H.; Wang, L.; Hollenberg, M.D. Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. Br. J. Pharmacol. 1996, 118, 521–530. [CrossRef] 73. Kong, W.; McConalogue, K.; Khitin, L.M.; Hollenberg, M.D.; Payan, D.G.; Böhm, S.K.; Bunnett, N.W. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2. Proc. Natl. Acad. Sci. USA 1997, 94, 8884–8889. [CrossRef][PubMed] 74. D’Andrea, M.R.; Rogahn, C.J.; Andrade-Gordon, P. Localization of protease-activated receptors-1 and -2 in human mast cells: Indications for an amplified mast cell degranulation cascade. Biotech. Histochem. 2000, 75, 85–90. [CrossRef][PubMed] 75. Nystedt, S.; Ramakrishnan, V.; Sundelin, J. The Proteinase-activated Receptor 2 Is Induced by Inflammatory Mediators in Human Endothelial Cells: Comparison with the thrombin receptor. J. Biol. Chem. 1996, 271, 14910–14915. [CrossRef][PubMed] 76. Abraham, L.A.; Chinni, C.; Jenkins, A.L.; Lourbakos, A.; Ally, N.; Pike, R.N.; Mackie, E.J. Expression of protease-activated receptor-2 by osteoblasts. Bone 2000, 26, 7–14. [CrossRef] 77. Xiang, Y.; Masuko-Hongo, K.; Sekine, T.; Nakamura, H.; Yudoh, K.; Nishioka, K.; Kato, T. Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β. Osteoarthr. Cartil. 2006, 14, 1163–1173. [CrossRef] 78. Palmer, H.S.; Kelso, E.B.; Lockhart, J.C.; Sommerhoff, C.P.; Plevin, R.; Goh, F.G.; Ferrell, W.R. Protease-activated receptor 2 mediates the proinflammatory effects of synovial mast cells. Arthritis Rheum. 2007, 56, 3532–3540. [CrossRef] 79. Boileau, C.; Amiable, N.; Martel-Pelletier, J.; Fahmi, H.; Duval, N.; Pelletier, J.-P. Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: A basic science study. Arthritis Res. Ther. 2007, 9, R121. [CrossRef] 80. Alier, K.A.; Endicott, J.A.; Stemkowski, P.L.; Cenac, N.; Cellars, L.; Chapman, K.; Andrade-Gordon, P.; Vergnolle, N.; Smith, P.A. Intrathecal Administration of Proteinase-Activated Receptor-2 Agonists Produces Hyperalgesia by Exciting the Cell Bodies of Primary Sensory Neurons. J. Pharmacol. Exp. Ther. 2007, 324, 224–233. [CrossRef] 81. Bohm, S.K.; Kong, W.; Bromme, D.; Smeekens, S.P.; Anderson, D.C.; Connolly, A.; Kahn, M.; Nelken, N.A.; Coughlin, S.R.; Payan, D.G.; et al. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem. J. 1996, 314 Pt 3, 1009–1016. [CrossRef] 82. Belham, C.M.; Tate, R.J.; Scott, P.H.; Pemberton, A.D.; Miller, H.R.P.; Wadsworth, R.M.; Gould, G.W.; Plevin, R. Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. Biochem. J. 1996, 320, 939–946. [CrossRef][PubMed] 83. DeFea, K.A.; Zalevsky, J.; Thoma, M.S.; Dery, O.; Mullins, R.D.; Bunnett, N.W. β-Arrestin–Dependent Endocytosis of Proteinase- Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2. J. Cell Biol. 2000, 148, 1267–1282. [CrossRef] [PubMed] 84. Seatter, M.J.; Drummond, R.; Kanke, T.; Macfarlane, S.R.; Hollenberg, M.D.; Plevin, R. The role of the C-terminal tail in protease- activated receptor-2-mediated Ca2+ signalling, proline-rich tyrosine kinase-2 activation, and mitogen-activated protein kinase activity. Cell. Signal. 2003, 16, 21–29. [CrossRef] 85. Ricks, T.K.; Trejo, J.A. Phosphorylation of Protease-activated Receptor-2 Differentially Regulates Desensitization and Internaliza- tion. J. Biol. Chem. 2009, 284, 34444–34457. [CrossRef] 86. Jung, S.-R.; Seo, J.B.; Deng, Y.; Asbury, C.L.; Hille, B.; Koh, D.-S. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling. J. Gen. Physiol. 2016, 147, 255–271. [CrossRef] 87. Roosterman, D.; Schmidlin, F.; Bunnett, N.W. Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2. Am. J. Physiol. Physiol. 2003, 284, C1319–C1329. [CrossRef][PubMed] 88. Smith, R.; Ransjö, M.; Tatarczuch, L.; Pagel, C.N.; Morrison, J.R.; Pike, R.N.; Mackie, E.J.; Song, S.-J. Activation of Protease- Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation. J. Bone Miner. Res. 2003, 19, 507–516. [CrossRef] 89. Georgy, S.R.; Pagel, C.N.; Ghasem-Zadeh, A.; Zebaze, R.M.D.; Pike, R.N.; Sims, N.A.; Mackie, E.J. Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair. Bone 2012, 50, 704–712. [CrossRef][PubMed] 90. Amiable, N.; Tat, S.K.; Lajeunesse, D.; Duval, N.; Pelletier, J.-P.; Martel-Pelletier, J.; Boileau, C. Proteinase-activated receptor (PAR)-2 activation impacts bone resorptive properties of human osteoarthritic subchondral bone osteoblasts. Bone 2009, 44, 1143–1150. [CrossRef] 91. Tsai, S.-H.; Sheu, M.-T.; Liang, Y.-C.; Cheng, H.-T.; Fang, S.-S.; Chen, C.-H. TGF-β inhibits IL-1β-activated PAR-2 expression through multiple pathways in human primary synovial cells. J. Biomed. Sci. 2009, 16, 97. [CrossRef] 92. Huang, X.; Ni, B.; Xi, Y.; Chu, X.; Zhang, R.; You, H. Protease-activated receptor 2 (PAR-2) antagonist AZ3451 as a novel therapeutic agent for osteoarthritis. Aging 2019, 11, 12532–12545. [CrossRef] 93. Abe, K.; Aslam, A.; Walls, A.F.; Sato, T.; Inoue, H. Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts. Life Sci. 2006, 79, 898–904. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 9352 15 of 16

94. Sawamukai, N.; Yukawa, S.; Saito, K.; Nakayamada, S.; Kambayashi, T.; Tanaka, Y. Mast cell-derived tryptase inhibits apoptosis of human rheumatoid synovial fibroblasts via rho-mediated signaling. Arthritis Rheum. 2010, 62, 952–959. [CrossRef][PubMed] 95. Nakano, S.; Mishiro, T.; Takahara, S.; Yokoi, H.; Hamada, D.; Yukata, K.; Takata, Y.; Goto, T.; Egawa, H.; Yasuoka, S.; et al. Distinct expression of mast cell tryptase and protease activated receptor-2 in synovia of rheumatoid arthritis and osteoarthritis. Clin. Rheumatol. 2007, 26, 1284–1292. [CrossRef] 96. Crilly, A.; Burns, E.; Nickdel, M.B.; Lockhart, J.C.; Perry, M.E.; Ferrell, P.W.; Baxter, D.; Dale, J.; Dunning, L.; Wilson, H.E.; et al. PAR2expression in peripheral blood monocytes of patients with rheumatoid arthritis. Ann. Rheum. Dis. 2012, 71, 1049–1054. [CrossRef][PubMed] 97. Tindell, A.G.; Kelso, E.B.; Ferrell, W.R.; Lockhart, J.C.; Walsh, D.A.; Dunning, L.; McInnes, I.B. Correlation of protease-activated receptor-2 expression and synovitis in rheumatoid and osteoarthritis. Rheumatol. Int. 2011, 32, 3077–3086. [CrossRef][PubMed] 98. Kelso, E.B.; Ferrell, W.R.; Lockhart, J.C.; Elias-Jones, I.; Hembrough, T.; Dunning, L.; Gracie, J.A.; McInnes, I.B. Expression and proinflammatory role of proteinase-activated receptor 2 in rheumatoid synovium: Ex vivo studies using a novel proteinase- activated receptor 2 antagonist. Arthritis Rheum. 2007, 56, 765–771. [CrossRef][PubMed] 99. Yan, S.; Ding, H.; Peng, J.; Wang, X.; Pang, C.; Wei, J.; Wei, J.; Chen, H. Down-regulation of protease-activated receptor 2 ameliorated osteoarthritis in rats through regulation of MAPK/NF-κB signaling pathway in vivo and in vitro. Biosci. Rep. 2020, 40, 1–16. [CrossRef] 100. Ferrell, W.R.; Lockhart, J.C.; Kelso, E.B.; Dunning, L.; Plevin, R.; Meek, S.E.; Smith, A.J.; Hunter, G.D.; McLean, J.S.; McGarry, F.; et al. Essential role for proteinase-activated receptor-2 in arthritis. J. Clin. Investig. 2003, 111, 35–41. [CrossRef] 101. Lucena, F.; McDougall, J.J. Pain responses to protease-activated receptor-2 stimulation in the spinal cord of naïve and arthritic rats. Neurosci. Lett. 2020, 739, 135391. [CrossRef] 102. Borbély, É.; Sándor, K.; Markovics, A.; Kemény, Á.; Pintér, E.; Szolcsányi, J.; Quinn, J.P.; McDougall, J.J.; Helyes, Z. Role of capsaicin-sensitive nerves and tachykinins in mast cell tryptase-induced inflammation of murine knees. Inflamm. Res. 2016, 65, 725–736. [CrossRef] 103. McDougall, J.J.; McConnell, M.; Reid, A.R. Intracellular versus extracellular inhibition of calpain I causes differential effects on pain in a rat model of joint inflammation. Mol. Pain 2021, 17, 17448069211016141. [CrossRef] 104. Kelso, E.B.; Lockhart, J.C.; Hembrough, T.A.; Dunning, L.; Plevin, R.; Hollenberg, M.D.; Sommerhoff, C.P.; McLean, J.S.; Ferrell, W.R. Therapeutic Promise of Proteinase-Activated Receptor-2 Antagonism in Joint Inflammation. J. Pharmacol. Exp. Ther. 2005, 316, 1017–1024. [CrossRef] 105. Busso, N.; Frasnelli, M.; Feifel, R.; Cenni, B.; Steinhoff, M.; Hamilton, J.; So, A. Evaluation of protease-activated receptor 2 in murine models of arthritis. Arthritis Rheum. 2006, 56, 101–107. [CrossRef][PubMed] 106. Crilly, A.; Palmer, H.; Nickdel, M.B.; Dunning, L.; Lockhart, J.C.; Plevin, R.; McInnes, I.B.; Ferrell, W.R. Immunomodulatory role of proteinase-activated receptor-2. Ann. Rheum. Dis. 2012, 71, 1559–1566. [CrossRef] 107. Ferrell, W.R.; Kelso, E.B.; Lockhart, J.C.; Plevin, R.; McInnes, I.B. Protease-activated receptor 2: A novel pathogenic pathway in a murine model of osteoarthritis. Ann. Rheum. Dis. 2010, 69, 2051–2054. [CrossRef][PubMed] 108. Amiable, N.; Martel-Pelletier, J.; Lussier, B.; Tat, S.K.; Pelletier, J.-P.; Boileau, C. Proteinase-activated Receptor-2 Gene Disruption Limits the Effect of Osteoarthritis on Cartilage in Mice: A Novel Target in Joint Degradation. J. Rheumatol. 2011, 38, 911–920. [CrossRef][PubMed] 109. Jackson, M.T.; Moradi, B.; Zaki, S.; Smith, M.M.; McCracken, S.; Smith, S.M.; Jackson, C.J.; Little, C.B. Depletion of Protease- Activated Receptor 2 but Not Protease-Activated Receptor 1 May Confer Protection Against Osteoarthritis in Mice Through Extracartilaginous Mechanisms. Arthritis Rheumatol. 2014, 66, 3337–3348. [CrossRef][PubMed] 110. Huesa, C.; Ortiz, A.C.; Dunning, L.; McGavin, L.; Bennett, L.; McIntosh, K.; Crilly, A.; Kurowska-Stolarska, M.; Plevin, R.; Hof, R.V.; et al. Proteinase-activated receptor 2 modulates OA-related pain, cartilage and bone pathology. Ann. Rheum. Dis. 2015, 75, 1989–1997. [CrossRef] 111. Muley, M.M.; Krustev, E.; Reid, A.R.; McDougall, J.J. Prophylactic inhibition of neutrophil elastase prevents the development of chronic neuropathic pain in osteoarthritic mice. J. Neuroinflam. 2017, 14, 1–12. [CrossRef] 112. Kaneva, M.K.; Muley, M.M.; Krustev, E.; Reid, A.R.; Souza, P.R.; Dell’Accio, F.; McDougall, J.J.; Perretti, M. Alpha-1-antitrypsin reduces inflammation and exerts chondroprotection in arthritis. FASEB J. 2021, 35, e21472. [CrossRef] 113. Connolly, A.J.; Ishihara, H.; Kahn, M.L.; Farese, R.V.; Coughlin, S.R. Role of the thrombin receptor in development and evidence for a second receptor. Nature 1996, 381, 516–519. [CrossRef] 114. Ishihara, H.; Connolly, A.J.; Zeng, D.; Kahn, M.L.; Zheng, Y.W.; Timmons, C.; Tram, T.; Coughlin, S.R. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997, 386, 502–506. [CrossRef][PubMed] 115. Hansen, K.K.; Saifeddine, M.; Hollenberg, M.D. Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells. Immunology 2004, 112, 183–190. [CrossRef] 116. McLaughlin, J.N.; Patterson, M.M.; Malik, A.B. Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization. Proc. Natl. Acad. Sci. USA 2007, 104, 5662–5667. [CrossRef] 117. Bah, A.; Chen, Z.; Bush-Pelc, L.A.; Mathews, F.S.; Di Cera, E. Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4. Proc. Natl. Acad. Sci. USA 2007, 104, 11603–11608. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 9352 16 of 16

118. Lin, H.; Liu, A.P.; Smith, T.H.; Trejo, J.A. Cofactoring and Dimerization of Proteinase-Activated Receptors. Pharmacol. Rev. 2013, 65, 1198–1213. [CrossRef] 119. Mwirigi, J.; Kume, M.; Hassler, S.N.; Ahmad, A.; Ray, P.R.; Jiang, C.; Chamessian, A.; Mseeh, N.; Ludwig, B.P.; Rivera, B.D.; et al. A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist. J. Pain 2021, 22, 692–706. [CrossRef][PubMed] 120. Kahn, M.L.; Zheng, Y.-W.; Huang, W.; Bigornia, V.; Zeng, D.; Moff, S.; Farese, R.V., Jr.; Tam, C.; Coughlin, S.R. A dual thrombin receptor. Nature 1998, 394, 11–15. [CrossRef] 121. Xu, W.-F.; Andersen, H.; Whitmore, T.E.; Presnell, S.R.; Yee, D.P.; Ching, A.; Gilbert, T.; Davie, E.W.; Foster, D.C. Cloning and characterization of human protease-activated receptor 4. Proc. Natl. Acad. Sci. USA 1998, 95, 6642–6646. [CrossRef] 122. French, S.L.; Hamilton, J.R. Protease-activated receptor 4: From structure to function and back again. Br. J. Pharmacol. 2016, 173, 2952–2965. [CrossRef] 123. Rudinga, G.R.; Khan, G.J.; Kong, Y. Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy. Int. J. Mol. Sci. 2018, 19, 573. [CrossRef][PubMed] 124. Shapiro, M.J.; Weiss, E.J.; Faruqi, T.R.; Coughlin, S.R. Protease-activated Receptors 1 and 4 Are Shut off with Distinct Kinetics after Activation by Thrombin. J. Biol. Chem. 2000, 275, 25216–25221. [CrossRef][PubMed] 125. Covic, L.; Gresser, A.L.; Kuliopulos, A. Biphasic Kinetics of Activation and Signaling for PAR1 and PAR4 Thrombin Receptors in Platelets†. Biochemistry 2000, 39, 5458–5467. [CrossRef] 126. Kim, Y.; Ghil, S. Regulators of G-protein signaling, RGS2 and RGS4, inhibit protease-activated receptor 4-mediated signaling by forming a complex with the receptor and Gα in live cells. Cell Commun. Signal. 2020, 18, 1–13. [CrossRef][PubMed] 127. Cunningham, M.R.; McIntosh, K.A.; Pediani, J.D.; Robben, J.; Cooke, A.E.; Nilsson, M.; Gould, G.W.; Mundell, S.; Milligan, G.; Plevin, R. Novel Role for Proteinase-activated Receptor 2 (PAR2) in Membrane Trafﬁcking of Proteinase-activated Receptor 4 (PAR4). J. Biol. Chem. 2012, 287, 16656–16669. [CrossRef] 128. Arachiche, A.; De La Fuente, M.; Nieman, M.T. Calcium Mobilization and Protein Kinase C Activation Downstream of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated by PAR3 in Mouse Platelets. PLoS ONE 2013, 8, e55740. [CrossRef] 129. Arachiche, A.; Mumaw, M.M.; de la Fuente, M.; Nieman, M.T. Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin. J. Biol. Chem. 2013, 288, 32553–32562. [CrossRef] 130. Russell, F.A.; Veldhoen, V.E.; Tchitchkan, D.; McDougall, J.J. Proteinase-Activated Receptor-4 (PAR4) Activation Leads to Sensitization of Rat Joint Primary Afferents Via a Bradykinin B2 Receptor-Dependent Mechanism. J. Neurophysiol. 2010, 103, 155–163. [CrossRef] 131. Russell, F.A.; Zhan, S.; Dumas, A.; Lagarde, S.; Pouliot, M.; McDougall, J.J. The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation. Pain 2011, 152, 354–360. [CrossRef] 132. Asfaha, S.; Cenac, N.; Houle, S.; Altier, C.; Papez, M.D.; Nguyen, C.; Steinhoff, M.; Chapman, K.; Zamponi, G.W.; Vergnolle, N. Protease-activated receptor-4: A novel mechanism of inﬂammatory pain modulation. Br. J. Pharmacol. 2007, 150, 176–185. [CrossRef][PubMed]